The present invention relates to methods and compositions for treating and preventing hepatic fibrosis and cirrhosis as well as fatty liver.
Adenosine is a nucleoside with wide distribution in the body. Adenosine mediates a broad array of physiological responses, including central nervous system sedation, inhibition of platelet aggregation and vascular smooth muscle vasodilation. These effects occur largely through interaction of adenosine with one of four types of adenosine receptors.
Adenosine thus perhaps represents a general regulatory substance, since no particular cell type or tissue appears uniquely responsible for its formation. In this regard, adenosine is unlike various endocrine hormones. There is no evidence for storage and release of adenosine from nerve or other cells. Thus, adenosine is unlike various neurotransmitter substances.
Adenosine, like the prostaglandins, may be characterized as a physiological regulator. In both cases the enzymes involved in the metabolic formation are ubiquitous and appear to be responsive to alterations in the physiological state of the cell. Receptors for adenosine, like those for prostaglandins, are very widespread.
Adenosine receptors comprise a group of cell surface molecules that mediate the physiologic effects of adenosine. Recent reviews include Stiles, G. F., Trends in Pharmacol. Sci. 7:485-490 (1986); Ramkumar, V. et al., Prog. Drug Res. 32:195-247 (1988); Olah, M. E. et al., Anu. Rev. Physiol. 54:211-225 (1992); Stiles, G. L. J. Biol. Chem. 267:6451-6454 (1992); Jacobson, K. A. et al., J. Med. Chem. 35:407-422 (1992). This family of receptors was originally classified as P1 or P2 purinergic receptors, dependent upon their preferential interactions with adenosine (P1) or ATP (P2) (Burnstock et al., in Cell Membrane Receptors of Drugs and Hormones, Straub et al., eds., Raven Press, New York, 1978, pp. 107-118). The P1 sites were further subdivided into A1, A2A, A2B, and A3 adenosine receptors based on their differential selectivity of adenosine analogues and molecular structure (Van Calker, D. et al., J. Neurochem 33:999-1005 (1979); Londos, C. et al. Proc. Natl. Acad. Sci. USA 77:2552-2554 (1980); Ralevic et al., Pharmacological reviews 50(3):413-92 (1998); Poulsen et al., Bioorganic and Medicinal Chemistry 6(6):619-41 (1998); Khakh et al., Trends in Pharmacological Sciences19(2): 39-41 (1998)). The A1 adenosine receptor, which is inhibitory to adenylyl cyclase, exhibits the potency order (R)-PIA greater than NECA greater than (S)-PIA. The A2 adenosine receptor, which is stimulatory to adenylyl cyclase, has a different potency order were NECA greater than (R)-(PIA) greater than (S)-PIA. ((R)-PIA is N6-phenylisopropyladenosine; (S)-PIA is N6-(S)-phenylisopropyladenosine; NECA is N-ethyl adenosine-5xe2x80x2-uronic acid). Both the A1 and the A2 adenosine receptors are widely distributed in the central nervous system and peripheral tissues (Ramkumar, V. et al., supra).
Additional information on adenosine receptors can be found in Ralevid et al., Pharmacological reviews 50(3):413-92, 1998; Poulsen et al., Bioorganic and Medicinal Chemistry 6(6):619-41, 1998 and Khakh et al., Trends in Pharmacological Sciences 19(2):39-41, 1998.
Four different adenine receptors have been claimed and their sequence is known. Until relatively recently, no truly useful radio ligand was available for characterizing the A2 adenosine receptors. Demonstration of adenosine receptors in smooth muscle was made primarily by functional assays, for example, adenosine stimulation of adenylyl cyclase activity via A2 receptors in vascular smooth muscle cells in culture (Anand-Srivastava, M. B. et al., Biochem. Biophys. Res. Commun. 108: 213-219 (1982); Anand-Srivastava , M. B. et al., Life Sci. 37: 857-867 (1985)). However, the concentrations of adenosine required to elevate cAMP were higher than those required of full vasorelaxation in vivo (Berne, R. M., Circ. Res. 47:807-813 (1980); Herlihy, J. T. et al., Am. J. Physiol. 230:1239-1243 (1976)). One cell line which has proved useful of studying A1 and A2 adenosine receptors (Ramkumar, V. et al., Molec. Pharmacol. 37:149-156 (1990) is the DDT1 MF-2 line, a smooth muscle cell line derived from a steroid-induced leimyosacroma of the vas deferens of an adult Syrian hamster (Norris, J. S. et al., Nature 248:422-424 (1974)).
Recently, two compounds were found to possess selective high affinity against radio ligands of the A2 receptor: [3H]CGS 21680 (Jarvis, M. R. et al., J. Pharmacolo. Exp. Their 251:888-893 (1989) and 125I-PAPA-APEC, the full chemical name of which is {2-3(2(2-(4-amino phenyl)methyl carbonyl-amino)ethylaminocarbonyl)ethyl]phenyl}ethylamino-5xe2x80x2-N-ethylcarboxamindoadenosine (Barrington, W. W. et al., Proc. Natl. Acad. Sci. USA 86:6572-6576 (1989)). Use of such ligands allowed identification of the A2 binding subunit as a 45 kDa protein (on SDS-PAGE) that was clearly distinguishable from the 38 kDa A1 binding subunit. Use of the azide derivative of 125-I-PAPA-APEC, a direct photo affinity probe of the A2 receptor, made it possible to demonstrate that the A2 binding subunit is a glycoprotein clearly different from the A1 receptor glycoprotein (Barrington, W. W. et al., Mol. Pharmacol. 38:177-183 (1990)). The A2 adenosine receptor has a single carbohydrate chain of either the complex or high mannose type.
Useful adenosine receptor agonists, in particular those with selectivity of the A2 receptor, are well known in the art. These include 2-substituted adenosine-5xe2x80x2-carboxamide derivatives (Hutchison, U.S. Pat. Nos. 4,968,697 and 5,034,381) and N9-cyclopentyl-substituted adenine derivatives (Chen et al., U.S. Pat. No. 5,063,233). These patents are hereby incorporated by reference in their entireties.
Adenosine and its analogues interact with neutrophils in inflammatory responses. While neutrophils are essential for limiting the spread of infection by a variety of microbes, stimulated neutrophils may damage injured tissues while en route to sites of infection or inflammation. Release of adenosine is one mechanism by which normal cells may protect themselves from activated neutrophils. Thus, one important action of adenosine and its analogues is the inhibition of generation of toxic oxygen products, including O2xe2x88x92 and H2O2, by interacting with A2 receptors on the neutrophil (Cronstein, B. N. et al., J. Immunol. 135:1366-1371 (1985); Roberts, P. A. et al., Biochem. H. 227:669-674 (1985); Schrier, D. J. et al., J. Immunol. 137:3284-3289 (1989); Iannone, M. A. et al., Fed. Proc. 44:580 (abstr.) (1985)). Adenosine promotes neutrophil chemotaxis via the A1 receptor (Cronstein, B.Nalet supra; Rose, F. R. et al., J. Exp. Med. 167:1186-1194 (1989)). Adenosine receptor ligation regulates inflammatory responses of neutrophils triggered by immune complexes acting through the Fcxcex3 receptor (Salmon, J. E., Immuno. 145:2235-2240 (1990)). Specifically, activation of A2 receptors inhibited these inflammatory responses, whereas activation of A1 receptors was stimulatory. These authors noted an important role of adenosine at picomolar concentrations as a promoter, and at micro molar concentrations as an inhibitor, of neutrophil responses elicited by immune complexes.
Interestingly, the immunosuppressive drug methotrexate, at low concentrations, acts as an anti-inflammatory agent at least in part due to its capacity to induce adenosine release by connective tissue cells such as dermal fibroblasts or umbilical vein endothelial cells. The released adenosine interacted with the neutrophil adenosine receptors (Cronstein, B. N. et al., Proc. Natl. Sci. USA 88:2441-2445 (1991)).
The nonselective adenosine receptor agonist, 2-chloroadenosine, inhibited adherence of stimulated neutrophils to endothelium, thus protecting the endothelium from inflammatory effects (Cronstein, B. N. et al., J. Clin, Invest. 78:760-770 (1986)). More recently, work from the present inventors"" laboratory has demonstrated that occupancy of A2 receptors inhibits neutrophil adherence and generation of toxic metabolites, thus contributing to an anti-inflammatory function (Cronstein, B. N. et al. J. Immunol. 148:2201-2206 (1992)).
The present inventors have thus found that adenosine regulates the accumulation of neutrophils at sites of inflammation. While neutrophils traverse through acellular connective tissue, the low concentrations of adenosine present promote phagocytosis, migration, and adherence to some, but not other, surfaces. Near foci of tissue injury, damaged cells release higher concentrations of adenosine that inhibit neutrophil adherence to cells and connective tissue substrata as well as inhibiting production of toxic oxygen metabolites by stimulated neutrophils. Thus, adenosine may promote accumulation of neutrophils at sites of tissue injury or microbial invasion, a pro-inflammatory function (Cronstein et al., 1992, supra)
It has been demonstrated that adenosine A2A receptor agonists promote wound healing. Enhanced dermal wound healing is accompanied by increased matrix (collagen) in the wounds. Cronstein et al., in U.S. Pat. No. 5,932,558, the entire contents of which are hereby incorporated by reference, disclose the use of adenosine receptor agonists for promoting wound healing. Increased adenosine release mediates many of the anti-inflammatory effects of methotrexate treatment, and the present inventors investigated whether methotrexate-stimulated adenosine release might also contribute to the methotrexate-induced hepatic fibrosis that occurs in a small number of patients.
A number of epidemiological studies have demonstrated that coffee consumption protects from development of cirrhosis: Lepore et al., xe2x80x9cThe Effect of Drinking Coffee and Smoking Cigarettes on the Risk of Cirrhosis Associated with Alcohol Consumption,xe2x80x9d European Journal of Epidemiology 10(6):657-664, 1994; Klatsky et al., xe2x80x9cCoffee, Tea and Immortality,xe2x80x9d Annals of Epidemiology 3(4):375-381, 1993; Klatsky et al., xe2x80x9cAlcohol, Smoking, Coffee, and Cirrhosis,xe2x80x9d American Journal of Epidemiology 136(10):1248-1257, 1992; Tanaka et al., xe2x80x9cCoffee Consumption and Decreased Serum Gamma-Glutamyltransferase and Aminotransferase Activities Among Male Alcohol Drinkers,xe2x80x9d International Journal of Epidemiology 27(3):438-443, 1998.
Caffeine, an ingredient in coffee, is a relatively non-selective adenosine receptor antagonist. The effects of caffeine on wakefulness, heart rate, etc., are all due to its capacity to block adenosine receptors. In recent epidemiological studies, coffee consumption appears to protect against the development of alcoholic cirrhosis, and one of the more prominent pharmacologic components of coffee is caffeine, a non-selective adenosine receptor antagonist (Lepore A. R. et al., European J. Epicemiol. 10(6):657-664 (1994).
Pharmacologically relevant concentrations of methotrexate and ethanol and combinations thereof caused increased adenosine release from HepG2 cells (a hepatoma cell line) in multiple experiments. The present inventors have observed in a number of experiments that CGS-21680, a relatively selective adenosine A2A receptor agonist, promotes collagen synthesis and release from a cultured rat stellate cell line in a dose-dependent fashion (EC50 approximately 300 nM) by as much as 20-fold (p less than 0.004). CSC, a specific adenosine A2A receptor antagonist almost completely blocks the CGS-21680-mediated promotion of collagen synthesis and release. DPCPX, an adenosine A1 receptor antagonist, and enprofylline, an A2B receptor antagonist, have little effect on the capacity of CGS-21680 to stimulate collagen release and synthesis.
Liang et al., in U.S. Pat. No. 5,859,019, describe methods for protecting against cardiac ischemia by administering adenosine A2A receptor antagonists, particularly 8-(3-chlorostyryl) caffeine, to patients suffering from ischemic damage or at risk for the same.
There is currently no treatment of the progression of development of liver fibrosis or cirrhosis or fatty liver other than antiviral therapy, which prevents underlying hepatic destruction.
It is an object of the present invention to overcome the aforesaid deficiencies in the prior art.
It is another object of the present invention to treat liver fibrosis, cirrhosis, or fatty liver.
It is a further object of the present invention to prevent liver fibrosis, cirrhosis, or fatty liver.
According to the present invention, agonists for adenosine A2A receptors promote synthesis of collagen by stellate cells. Antagonists at these receptors prevent the agonist-mediated increase in collagen synthesis. During tissue injury or necrosis, or after exposure to ethanol, the hepatocytes release high concentrations of adenosine, which may stimulate production of collagen in the liver, leading to hepatic fibrosis and cirrhosis. Thus, administration of adenosine A2 receptor antagonists can block promotion of collagen synthesis and release, and thus prevent and treat hepatic fibrosis, cirrhosis, or fatty liver.